Native plasma-derived FVIII/VWF complex has lower sensitivity to FVIII inhibitors than the combination of isolated FVIII and VWF proteins. Impact on Bethesda assay titration of FVIII inhibitors

Sensitivity to FVIII inhibitors of the native plasma-derived (pd) FVIII/VWF complex vs. the complexes formed after exogenous FVIII infusion in the haemophilic patient has not been thoroughly studied. The role of VWF in the interaction of FVIII with inhibitors was studied in vitro using different combinations of VWF and FVIII concentrates. Normal plasma, pdFVIII/VWF and isolated FVIII (recombinant FVIII, B-domain deleted and pdFVIII) were used. Titre (BU) was kinetically determined (up to 2 h) in serial dilutions of inhibitor IgG (purified from a pool of plasmas with inhibitors) mixed with VWF and then incubated with the different FVIII. Inhibitor was also added to previously mixed VWF+FVIII. Residual FVIII:C was determined. TGA assays were performed with FVIII-deficient plasma spiked with the FVIII-VWF mixtures with/without an ESH-8 antibody. Inhibitor titres for plasma and pdFVIII/VWF were comparable at all time points. Titres for all concentrates of isolated FVIII were significantly higher than those for plasma or pdFVIII/VWF (1.4-1.9 fold) even after preincubation with VWF. At t = 0 h, titres for plasma or pdFVIII/VWF were unquantifiable, but were detectable for isolated FVIII (0.6-1.6 BU). In contrast to pdFVIII/VWF, the decrease in thrombin generation parameters by isolated FVIII in the presence of ESH-8 was significant (P < 0.01) even when previously combined with VWF. In conclusion, VWF protection against FVIII inhibitor activity might be higher with native pdFVIII/VWF complex than with the corresponding compound formed from the isolated proteins. Bethesda assay titration using different FVIII concentrates would be advisable to guide the treatment of inhibitor patients.